Leishmaniasis is a major health problem in humans and is caused by the protozoan parasite Leishmania. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Our laboratory has focused on metabolic pathways identified through the study of antifolate-resistant Leishmania parasites, which has lead us into studies of the metabolism of the unconjugated pteridine biopterin and the conjugated pteridine folate, and the development and application of new genetic tools. Studies of biopterin metabolism have revealed roles in growth, differentiation to infective forms in vitro and in the sand fly, virulence in animal infections, and in a novel pathway of oxidant resistance and susceptibility. This in turn has led to the discovery and study of a new family of pteridine binding proteins potentially playing key roles linking many of these biopterin-dependent activities. Inhibition studies using a panel of novel antifolates have revealed new approaches to parasite chemotherapy. Here we describe results from a systematic analysis of the Leishmania folate metabolic pathway, comprising interconversions of 1-carbon tetrahydrofolate metabolites needed to make essential metabolites in intermediary metabolism including DNA, RNA, and amino acids. Our studies have disclosed an unanticipated richness in the folate pathway, revealing an unanticipated divergence from that of the mammalian host. These studies have yielded new opportunities for chemotherapeutic attack and new directions for future study of pteridine metabolism and biology. The three specific aims of this competing renewal application are: 1. To characterize the role of an essential cofactor, 10-formyl tetrahydrofolate (10-CHO-THF) in Leishmania metabolism and biology, and to study the enzyme 5,10-methylene tetrahydrofolate dehydrogenase/cyclohydrolase (DHCH1) primarily responsible for its synthesis. Since DHCH1 is an essential enzyme, these studies will make good use of two new innovative approaches in Leishmania, a system for regulated protein expression and plasmid 'shuffling'methods. 2. To determine whether Leishmania's dependency on 10-CHO-THF arises through its role in critical steps of mitochondrial protein synthesis. This will be probed by studies of the enzyme methionyl-tRNA formyltransferase (FMT), which uses 10-CHO-THF to formylate Methionyl-tRNAMet required for initiation of protein synthesis in that compartment. 3. To characterize the novel pteridine-binding protein YGFZ, defining its activities and its specific pteridine binding partners, and to characterize its role in Leishmania biology and virulence. YGFZ has been implicated in a number of important process including oxidant resistance and the formation of critical Fe/S clusters, and its diverse activities may provide a 'missing link'for the diverse activities o pteridines in many aspects of Leishmania biology. PUBLIC HEALTH RELEVANCE: Leishmania are important tropical parasites, causing disease in more than 10 million people worldwide;more than 400 million people are at risk for infection in endemic regions. US military personnel have significant risk of infection in these areas as well, and recently concerns have risen that leishmaniasis may be endemic in certain dog populations in the USA. Depending on the species, Leishmania-induced pathology ranges from self-healing, cutaneous lesions to fatal, visceral diseases. Currently, there are no vaccines available against leishmaniasis, and the only approved chemotherapies are marginally effective, difficult to administer, and have significant associated toxicities. The underlying tenet of our research program is that improved understanding of key pathways required for parasite virulence and viability may provide opportunities for the development of improved therapies. Our laboratory has focused on metabolic pathways identified through the study of antifolate-resistant Leishmania parasites, which has lead us into studies of the metabolism of the unconjugated pteridine biopterin and the conjugated pteridine folate, and the development and application of new genetic tools required to answer key questions. Studies of biopterin metabolism have revealed a requirement for growth, a role in controlling parasite differentiation in vitro and in the sand fly, a requirement for virulence in animal infections, and implicated reduced biopterin in a novel pathway of oxidant resistance and susceptibility. This in turn has led to the studies of a new family of pteridine binding proteins, which potentially may provide the 'missing'link in our understanding of how biopterin mediates all its important activities. Inhibition studies using a panel of novel antifolates and parasites specifically lacking or overproducing pteridine or folate reductases have revealed two new approaches to parasite chemotherapy, one involving combined inhibition of PTR1 and DHFR, and a second implicating a new and as yet unidentified target. The combined inhibition approach has been validated by our own and recent studies by others. Here we describe a systematic analysis of the Leishmania folate metabolic pathway. This pathway consists of a series of metabolic interconversions of the reduced form of the vitamin folate acid, involving transfers of one-carbon building blocks needed to make essential metabolites in intermediary metabolism including DNA, RNA, and amino acids. A fruitful series of gene by gene reverse genetic approaches has disclosed an unanticipated richness in the folate metabolic pathway, with unanticipated divergence from that of the mammalian host. These studies have yielded new opportunities for chemotherapeutic attack and new directions for future study of pteridine metabolism and biology. [unreadable]